Cell Chemical Biology
Volume 27, Issue 10, 15 October 2020, Pages 1241-1249.e4
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Article
Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design

https://doi.org/10.1016/j.chembiol.2020.07.021Get rights and content
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Highlights

  • Searched for synthetic ligands to the ZTP riboswitch using two approaches

  • Identified hits that replace the natural ligand's ribose moiety with a pyridine

  • Lead compound binds to and activates ZTP riboswitches in vitro and in vivo

  • Co-crystal structures suggest affinity is achieved via π-π stacking interactions

Summary

Riboswitches are mRNA domains that make gene-regulatory decisions upon binding their cognate ligands. Bacterial riboswitches that specifically recognize 5-aminoimidazole-4-carboxamide riboside 5′-monophosphate (ZMP) and 5′-triphosphate (ZTP) regulate genes involved in folate and purine metabolism. Now, we have developed synthetic ligands targeting ZTP riboswitches by replacing the sugar-phosphate moiety of ZMP with various functional groups, including simple heterocycles. Despite losing hydrogen bonds from ZMP, these analogs bind ZTP riboswitches with similar affinities as the natural ligand, and activate transcription more strongly than ZMP in vitro. The most active ligand stimulates gene expression ∼3 times more than ZMP in a live Escherichia coli reporter. Co-crystal structures of the Fusobacterium ulcerans ZTP riboswitch bound to synthetic ligands suggest stacking of their pyridine moieties on a conserved RNA nucleobase primarily determines their higher activity. Altogether, these findings guide future design of improved riboswitch activators and yield insights into how RNA-targeted ligand discovery may proceed.

Keywords

RNA
structure
kinetic control
target-based design
transcription
X-ray crystallography
small molecule
microarray

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